**3.2 Process microbiology**

The anaerobic degradation of complex organic matter is carried out by different groups of bacteria as indicated in Fig. 1. These exists a coordinated interaction among these bacteria. All process may fail if one group is inhibited (Khanal, 2008).

from H2 and CO2 by the hydrogenotrophic methanogens (group 4). Since methane is largely generated from acetate, acetotrophic methanogenesis is the rate-limiting step in anaerobic wastewater treatment. The synthesis of acetate from H2 and CO2 by homoacetogens (group 5) has not been widely studied. Mackie and Bryant (1981) reported that acetate synthesis through this pathway accounts for only 1-2% of total acetate formation at 40°C and 3-4%

> **Polymers**  Proteins, polysaccharides, lipids

> > **Monomers y oligomers**  Amino acids, sugars, Fatty acids

**1**

**1**

*i) Hydrolysis*

*ii) Fermentation* 

Fig. 1. Steps of anaerobic digestion of complex organic matter (the number indicate the

CH4 + CO2

The anaerobic degradation of complex organic matter is carried out by different groups of bacteria as indicated in Fig. 1. These exists a coordinated interaction among these bacteria.

H2 + CO2 Acetate

*iv) Methanogenesis*

**3**

**5 4**

*iii) Acetogenesis*

**2**

**Intermediaries** Propionate, butyrate, alcohols

**1 1**

total solids at 60°C in a cattle waste digester.

group of bacteria involved in the process).

All process may fail if one group is inhibited (Khanal, 2008).

**3.2 Process microbiology** 


CO2 + H2 CH3COOH + 2H2O (1)

$$\text{4CO} + \text{2H}\_2\text{O} \rightarrow \text{CH}\_3\text{COOH} + \text{2CO}\_2\tag{2}$$

$$4\text{HCOOH} \rightarrow \text{CH}\_3\text{COOH} + 2\text{CO}\_2 + 2\text{H}\_2\text{O} \tag{3}$$

$$\text{4CH}\_3\text{OH} + 2\text{CO}\_2 \rightarrow \text{3CH}\_3\text{COOH} + 2\text{CO}\_2\tag{4}$$

*Acetobacterium woodii* and *Clostridium aceticum* are the two mesophilic homoacetogenic bacteria isolated from sewage sludge (Novaes1986). Homoacetogenic bacteria have a high thermodynamic efficiency; as result there is no accumulation of H2 and CO2 during growth on multicarbon compounds (Zeikus 1981).

d. **Metanogenic Bacteria (group 4 and 5):** Methanogens are obligate anaerobes and considered as a rate-limiting specie in anaerobic treatment of wastewater. Abundant methanogens are found in anaerobic environments rich in organic matter such as swamps, marches, ponds, lake and marine sediments, and rumen of cattle. Most methanogens can grow by H2 as a source of electrons via hydrogenase as shown in the follow reaction (Eq. 5) (Khanal, 2008):

$$4\text{H}\_2 + \text{CO}\_2 \rightarrow \text{CH}\_4 + 2\text{H}\_2\text{O}\tag{5}$$

The source of H2 is the catabolic product of other bacteria in the system, such as hydrogenproducing fermentative bacteria, especially *Clostridia* (group 1) and hydrogen-producing acetogenic bacteria (group 2). The hydrogenotrophic pathway contributes up to 28% of the

Biogas Production from Anaerobic Treatment of Agro-Industrial Wastewater 97

(Fannin, 1987). In the natural environments, the optimum temperature for the growth of methane forming *archaea* is 5-25 ºC for psychrophilic, 30-35 ºC, for mesophilic, 50-60 ºC, for

It is generally understood that higher temperature could produce higher rate of reaction and thus promoting higher application of organic loading rate (OLR) without affecting the organic removal efficiency (Chae et al., 2007; Choorit and Wisarnwan, 2007; Poh and Chong, 2009). Using palm oil mill effluent as the substrate, Choorit and Wisarnwan (2007) demonstrated that when the digester was operated at thermophilic temperature (55 ºC), showed higher OLR application than the that of mesophilic (17.01 against 12.25 g COD/ m3-d) and the methane productivity was also higher (4.66 against 3.73 L/L/d) (Choorit and Wisarnwan, 2007). A similarly study by Chae et al (2007), indicated that the higher temperature of 35 ºC led to the highest methane yield as compared to 30 ºC and 25 ºC

Using cheese whey, poultry waste and cattle dung as substrates, Desai et al. (1994) showed that when the temperature was increased from 20, 40 and 60 ºC, the biogas production and methane percentage increased as well. The digestion rate temperature dependence can be

where *t* is temperature in ºC, and *rt*, *r30* are digestion rates at temperature *t* and 30ºC, respectively. Based in Eq. 7, the decrease in digestion rate for each 1 ºC decreased in temperature below the optimum range is 11%. Similarly, the calculated rate at 25 ºC y 5 ºC

Although the thermophilic anaerobic process could increase the rate of reaction, the yield of methane that could be achieved over the specified organic amount is the same regardless of the mesophilic or thermophilic conditions. That value is 0.25 kg CH4/kg COD removed or 0.35 m3 CH4/kg COD removed (0 ºC, 1 atm) which is derived by balancing the following equation (Eq. 8), taking into account the different operating conditions worked, can be explained that the values obtained for methane production is different in many scientific

 CH4+2O2 CO2+2H2O (8) Although thermophilic condition could result in higher application of organic loading rates and better destruction of pathogens, at the same time it is more sensitive to toxicants and temperature control is more difficult (Gerardi, 2003; Choorit and Wisarnwan, 2007). Furthermore, biomass washout that could lead to volatile fatty acids accumulation and methanogenesis inhibition could also occur if the thermophilic temperature could not be controlled (Poh and Chong, 2009). As a result, in tropical regions mesophilic temperatures are the preferred choice for anaerobic treatment (Yacob et al., 2005, Sulaiman et al., 2009).

As far as the anaerobic digestion process is concerned, it is more appropriate to discuss alkalinity and pH together because these parameters are related to each other and very

are 59 and 7% respectively, relative to the rate at 30 ºC (Dasai et al., 1994).

�� � ���(1.11)(����) (7)

thermophilic and >65 ºC for hyprethermophilic (Tchobanoglous and Burton, 1996).

although the methane contents only changed slightly.

expressed using Arrhenius expression:

reports:

**4.2 Alkalinity and pH** 

methane generation in an anaerobic treatment system. It bears mentioning that there are many H2-using methanogens that can use formate as a source of electrons for the reduction de CO2 to methane, as show in reaction (Eq. 6):

$$4\text{HCOO} + 2\text{H}^\* \rightarrow \text{CH}\_4 + \text{CO}\_2 + 2\text{HCO}\_3^\cdot \tag{6}$$
